PROJECT SUMMARY Histone post-translational modifications (PTMs) affect a variety of nuclear processes, including gene regulation, DNA repair, and maintenance of chromatin structure. Misregulation of these chemical modifications has broad implications for human biology and drives a myriad of genetic diseases. While most PTMs are small chemical moieties (e.g. methylation and acetylation), monoubiquitination entails the covalent attachment of the bulky 8.5 kDa protein ubiquitin to the side-chain amino group of target lysines. Ubiquitination of histone proteins H2A (H2Aub) and H2B (H2Bub) serves important roles in DNA damage repair and transcriptional regulation. Indeed, H2Aub and H2Bub, along with the enzymes associated with the addition and removal of ubiquitin, have been implicated in various cancers. Preliminary experiments show strong evidence that the ubiquitin moiety itself on both H2Aub and H2Bub contains numerous methylation and acetylation PTMs in vivo. Hereafter, these modifications are called ?second-tier? PTMs to distinguish them from the many examples of PTMs that are directly conjugated to histone proteins. This proposed work pursues the hypothesis that second-tier PTMs contribute to and alter the functional output of chromatin ubiquitination of histones H2A and H2B, ultimately affecting chromatin structure, gene activation, and gene repression. The goals of the proposed research are to (1) validate the second-tier PTMs on H2Aub and H2Bub identified in preliminary experiments using alternative approaches, (2) determine where these modifications are distributed over the epigenome and whether these modifications are altered as a function of cell state and type, (3) identify the enzymes that install these PTMs, and (4) investigate the functional role of these modifications in chromatin biology. Central to these endeavors will be the core expertise of Professor Tom Muir?s lab in producing ?designer chromatin,? a process that uses chemical biology, protein engineering, and chromatin reconstitution techniques to create chemically-defined nucleosomes. Designer chromatin will be used in combination with biochemical, genetic, and proteomics tools to accomplish the proposed research goals. These experiments will elucidate the functional role of this unexplored level of chromatin modification and deepen our fundamental understanding of histone ubiquitination in chromatin. In addition, the proposed research will develop a methodological and experimental framework for studying second-tier PTMs, which will guide future efforts to study these modifications, whether in the context of chromatin or other cytosolic processes.